Date Published: March 20, 2014
Publisher: Public Library of Science
Author(s): Gavin J. Wright, Julian C. Rayner.
Throughout their extraordinarily complex life cycle, Plasmodium parasites must navigate a wide range of intracellular and extracellular environments in both vertebrates and invertebrates. To achieve this, the parasite develops into a series of distinct morphological forms or “zoites,” each of which is specialised for a particular biological challenge. Merozoites—ovoid cells approximately 1 µm long that are released from an infected erythrocyte once development is complete—are the epitome of a specialised Plasmodium stage. Merozoites do not replicate outside of their host: they exist purely to find and invade erythrocytes. To do so, they undergo a series of complex manoeuvers, first visualised by pioneering video microscopy and electron microscopy studies more than 30 years ago , . Initial contacts between the merozoite and erythrocyte can occur at any point on the merozoite surface, which are rapidly followed by the reorientation of the polar merozoite such that its apical end directly apposes the erythrocyte membrane (see Figure 1). This allows the parasite to deploy a series of specialised apically located secretory organelles: rhoptries, micronemes, and dense granules. These organelles then discharge their contents in a regulated and ordered schedule during and immediately after the invasion process at the site of contact –. Ligands released in this manner interact with erythrocyte surface receptors to form an electron-dense thickening of the erythrocyte membrane at the nexus of erythrocyte–merozoite contact. The junction is passed around the merozoite surface in a belt-like structure, driven by an actin-myosin motor that is anchored to the merozoite’s inner membrane complex (IMC), which contributes to the formation and maintenance of the merozoite’s characteristic ovoid shape , . Invasion is completed as the moving junction closes behind the merozoite in the fashion of an iris diaphragm, leaving the merozoite enclosed within a parasitophorous vacuole.
Given the complexity of the invasion process, it is no surprise that the merozoite expresses a diverse array of invasion-associated proteins. The combination of genome sequencing , large-scale gene ,  and protein profiling studies , , together with the rapid expansion in P. falciparum–experimental genetic technologies  have identified more than 50 P. falciparum proteins that are hypothesised to somehow be involved in the invasion process, although in the vast majority of cases their precise function is unknown. The most well-studied of these have been organised into distinct functional classes: MSPs (merozoite surface proteins), which form a structurally complex coat around the merozoite surface, and the PfEBAs (P. falciparum erythrocyte binding antigens, related to the P. vivax duffy binding protein) and PfRHs (P. falciparum reticulocyte binding protein [RBP] homologues, related to the P. vivax RBPs [PvRBPs]), which are stored in specialised apical organelles, the rhoptries and micronemes , .
Not only are MSPs often highly polymorphic, making them challenging targets, they also generally have poorly defined functions. A more rational approach would be to use a mechanistic understanding of the parasite and host molecules involved in invasion to identify targets for a potential invasion-blocking therapeutic. For many years, invasion research has focused on the role of the PfRHs and PfEBAs, and this work has led to significant advances in mechanistic understanding , . However, the potential of PfRHs and PfEBAs as intervention targets has been compounded by another evasion mechanism used by the parasite—functional redundancy. It has been known for some time that P. falciparum merozoites can use several alternative pathways to invade human erythrocytes. The definition of what exactly constitutes an “alternative invasion pathway” is not clear, and the area in general is in urgent need of a systematic overhaul and agreement on terminology. A simple and pragmatic definition is that when the repertoire of available erythrocyte receptors is restricted in vitro either by enzyme treatment (generally with trypsin, chymotrypsin, or neuraminidase) or through the use of erythrocytes from human donors with defined blood groups, there can be a range of phenotypic outcomes depending on the P. falciparum strain. Culture-adapted P. falciparum strains have long been known to have differential abilities to invade both enzyme-treated erythrocytes  and erythrocytes from individuals that lack expression of specific surface receptors , . Similar observations have been made using field isolates, both in strains that have recently been adapted to in vitro culture , and in parasites that have never been adapted but were phenotyped in their first round of invasion in vitro , . A large body of experimental data suggests that it is the PfRH and PfEBA protein families that are responsible for this functional redundancy: when individual ligands in these families are genetically deleted, a change in the ability of the parasites to invade enzyme-treated erythrocytes is the most commonly observed phenotype –. Similar effects can be observed by the addition of antibodies directed to the RHs (reticulocyte binding protein homologues) or EBAs (erythrocyte binding antigens) in parasite growth assays –.
Are there any other invasion ligands that could be targeted that avoid the problem of PfRH and EBA redundancy? So far, there are two parasite ligands that can be targeted by antibodies to induce a potent block in invasion and also appear to be essential and nonredundant, as attempts to genetically delete them have failed. Both have known receptors: PfRH5 and its erythrocyte receptor, basigin, and AMA1 (apical membrane antigen) and its parasite-encoded receptor, RON2 (rhoptry neck protein) (see Figure 3).
Given that it is essential for the survival of blood-stage parasites, erythrocyte invasion has long been viewed as a point in the life cycle that could be rationally targeted in the development of an anti-malarial vaccine. Although vaccine development priorities have recently become focused primarily on transmission blocking and pre-erythrocytic stages, the development of the RTS,S vaccine reinforces the fact that vaccines directed at a single target are never likely to be 100% effective. Furthermore, a highly effective blood-stage vaccine will, by definition, affect transmission by reducing the pool of ring-stage parasites capable of gametocyte differentiation. It is our strong opinion that invasion targets must be considered as crucial components of any second-generation multistage malaria vaccine. However, one of the very features of invasion that make it an attractive vaccine target—its exposure to the antibody-mediated immune response—also makes it a difficult target, because the host–parasite “arms race” has forced the parasite to evolve sophisticated immunoprotective mechanisms to shield itself. In particular, the parasite has protected the MSPs and RH/EBA ligands by generating sequence diversity and functional redundancy, resulting in parasites that use experimentally definable alternative invasion pathways that are difficult to target. One possible way to circumvent this problem would be to generate a multicomponent vaccine that attempts to neutralise all alternative ligands, but this is likely to be expensive to manufacture, and at best may simply recapitulate the partial protection found in clinically immune adults. Nonredundant interactions essential for invasion (AMA1–RON2 and RH5–basigin) make conceptually more attractive targets, but perhaps unsurprisingly, the parasite has evolved mechanisms to protect these critical invasion ligands. Intriguingly, however, the parasite protects AMA1 and RH5 from the host immune response by different mechanisms: it protects AMA1 by creating a series of immunologically distinct variants, while native RH5 appears immunoprotected. Critically, RH5 does not appear to be intrinsically nonimmunogenic since high antibody titres to RH5 are readily obtained in experimental models, and can potently inhibit invasion in vitro , . This raises the possibility that the parasite’s immunoprotective mechanisms could be circumvented by eliciting unnatural immunity with an RH5-based vaccine. One of the primary challenges in RH5 vaccine development will clearly be identifying adjuvants that raise sufficiently high antibody titres in the absence of immune boosting. However given the lack of success to date in circumventing the merozoite’s immune evasion mechanisms, developing a potent anti-RH5 immune response and identifying other targets that functionally synergise with such responses represents the current best hope for an invasion-blocking vaccine.